The Protective Effect And Its Mechanism Of Aqueous Extract From Fomes Fomentarius Compound On Carbon Tetrachloride Induced Liver Injury

Objective:Establish the liver acute injury model by carbon tetrachloride in vitro and in vivo；Tostudy the protective effects of aqueous extract from Fomes fomentarius compound（AEFFC） on carbon tetrachloride induced liver injury and to explore its mechanism；Tostudy acute toxicity of AEFFC in the Mice，which could provide scientific evidence forfurther study and clinical use.Materials and methods:1. Protective effects of AEFFC on L-02cell line injury induced by CCl₄（1） To establish a stable liver injury model induced by CCl₄；（2）The cells were divided into different groups：CCl₄injured group（20mmol/L），AEFFC protective groups（50、100、200μg/mL），normal control group and VE group（50mmol/L）；（3）Cell viability was detected by MTT assay；（4）ALT、AST in the culture medium of L-02cells were measured via the biochemicalassay kits；（5）Intracellular MDA and SOD were measured via the biochemical assay kits；2. Protective effects of AEFFC on acute liver injury mice induced by CCl₄（1） To build mice acute liver injury models induced by CCl₄.The mice were randomlydivided into6groups，each group with10mice: control group, model group, AEFFCprotective groups（100、200、400mg/kg）, positive drug group（Bifendate150mg/kg）. Thesedrugs were administration orally for7days（once a day）.Acute liver injury mice wereinduced by intraperitoneal administration of0.1%CCl₄of peanut oil solution two hours after the last administration except the control group, and the control group was given byintraperitoneal administration of peanut oil solution；（2） ALT、AST and TNF-α in the serum were measured via the biochemical assay kits；（3） Histopathological changes were observed by staining with Haematoxylin-eosine；（4） Liver apoptotic index was examined by TUNEL in liver tissue；（5） The content of MDA and the activities of SOD in liver tissue were examined；（6） RT-PCR were used to detect the mRNA expression of TNF-α.3. Mice acute toxicity test of AEFFC40mice were randomly divided into the control group and the AEFFC group, eachgroup with20mice: the AEFFC group was given0.48g/ml AEFFC （the largestconcentration of dissolved） and the control group was given distilled water, twice per day.The active appearance of the mice and the number of deaths were observed and recordedfor14days. Multiples of tolerance were calculated using the formula.Results:1. Protective effects of AEFFC on L-02cells injury induced by CCl₄（1） With the increasing of concentration of CCl₄, the L-02cells survival rate decreasedin a dose-dependent manner．We establish a stable liver injury model induced by20mmol/L CCl₄；（2） Morphological observation：There were more cells undergo apoptosis in injurygroup cells，while the AEFFC protection groups cell survival status were superior to injurygroups in a dose-dependent manner．（3） Compared with normal group, ALT, AST levels of model group were significantlyhigher; Compared with model group, AEFFC groups could reduce the elevated ALT, ASTlevel.（4） Compared with normal group, MDA level of model group were significantlyhigher, meanwhile the activity of SOD were reduced, AEFFC（100、200μg/mL）couldreduce the levels of MDA as well as enhancing the activity of SOD；（5） MTT assay：cell viability in model group reduced markedly（55.4%），AEFFC canenhance cell viability evidently，and the protective effect enhanced with the increase ofconcentration of AEFFC； 2. Protective effects of AEFFC on acute liver injury mice induced by CCl₄（1） Compared with the control group, the liver index was5.12±0.41%and significantlyincreased in model group; AEFFC groups and Bifendate group were lower than that ofmodel group；（2） Compared with the control group, the level of ALT and AST significantlyincreased in model group; AEFFC groups and Bifendate group were lower than that ofmodel group；（3） Compared with the control group, the level of TNF-α significantly increased inmodel group; AEFFC groups were lower than that of model group；（4）Compared with the control group, the activity of SOD in liver tissue significantlydecreased in model group; AEFFC groups and Bifendate group were higher than that ofmodel group. Contrast with SOD,compared with control group, the content of MDA inliver tissue significantly increased in model group; AEFFC groups and Bifendate groupwere lower than that of model group；（5） Control group of mice visible to the naked eye was bright red in liver tissue withgood gloss, texture, soft and flexible. Liver size of model group was significantly increased,texture and hard. The sizes and colors of the liver of AEFFC groups and BDP group werebetween control group and model group. The result of Haematoxylin-eosine: The structureof hepatic lobules was clear and the structure of liver cell was normal in control group; Asto the model guoup, the structure of hepatic lobules was not clear; The liver cell wasswelling and focal necrosis. The degree of liver injury in treatment groups with AEFFCwas significantly reduced；（6） TUNEL staining showed that a lot of positive green nucleus were expressed andnuclear chromatin bordering in model group.Compared with control group, the apoptoticindex in liver of acute liver injury mice significantly increased in model group; AEFFCgroups and BDP group were lower than that of model group;（7） RT-PCR assay found that differences were’t detected in the expressions of TNF-α.3. Mice acute toxicity test of AEFFCThe MTD of mice was24g/Kg in the acute toxicity test of AEFFC,it showed thatAEFFC was safe and it had no adverse effect.Conclusions: This study indicates thatAEFFC has a distinct protective effect on liver injury inducedby CCl₄．The liver protective mechanism may relate to its abilities of eliminating freeradical and decreasing the levels of TNF-α in serum, and inhibit lipid peroxidation and cellapoptotic.